Restoration of spermatogenesis and fertility in azoospermic mutant mice by suppression and reelevation of testosterone followed by intracytoplasmic sperm injection.

Advances in assisted reproduction techniques such as in vitro fertilization and intracytoplasmic sperm injection have made paternity possible for many patients with male infertility. However, at least some sperm or spermatids are required for these techniques to be successful, and patients incapable of producing spermatids cannot be helped. Male mice homozygous for the mutant juvenile spermatogonial depletion (jsd) gene show spermatogonial arrest and an elevated intratesticular testosterone level like many other experimental infertility models such as those with iradiation- or chemotherapy-induced testicular damage. In this category of infertile males, suppression of the testosterone level induces spermatogonial differentiation to the stage of spermatocytes but no further. In the present study with jsd mutant mice, we induced spermatogenesis first to spermatocytes and then to elongated spermatids by suppression of testosterone levels with a GnRH antagonist, Nal-Glu, at a dose of 2500 microg kg(-1) day(-1) for 4 wk and then withdrawal of Nal-Glu. Spermatids were seen in the cross-sections of seminiferous tubules in all mice treated by administration and subsequent withdrawal of Nal-Glu. Four weeks after withdrawal of Nal-Glu, some of the germ cells differentiated into elongated spermatids. Supplementation with testosterone and Nal-Glu after 4 wk of treatment with Nal-Glu alone also induced spermatogenesis similar to the induction by withdrawal of Nal-Glu. Thus, we ascribe the restoration of the differentiation of spermatocytes to spermatids to reelevation of the testosterone level. Furthermore, we successfully rescued male sterility in jsd mice by subsequent intracytoplasmic sperm injection using the elongated spermatids induced by the programmed hormone therapy.     

Protective mechanism of reduced water against alloxan-induced pancreatic β-cell damage: Scavenging effect against reactive oxygen species

Reactive oxygen species (ROS) cause irreversible damage to biological macromolecules, resulting in many diseases. Reduced water (RW) such as hydrogen-rich electrolyzed reduced water and natural reduced waters like Hita Tenryosui water in Japan and Nordenau water in Germany that are known to improve various diseases, could protect a hamster pancreatic β cell line, HIT-T15 from alloxan-induced cell damage. Alloxan, a diabetogenic compound, is used to induce type 1 diabetes mellitus in animals. Its diabetogenic effect is exerted via the production of ROS. Alloxan-treated HIT-T15 cells exhibited lowered viability, increased intracellular ROS levels, elevated cytosolic free Ca²⁺ concentration, DNA fragmentation, decreased intracellular ATP levels and lowering of glucose-stimulated release of insulin. RW completely prevented the generation of alloxan-induced ROS, increase of cytosolic Ca²⁺ concentration, decrease of intracellular ATP level, and lowering of glucose-stimulated insulin release, and strongly blocked DNA fragmentation, partially suppressing the lowering of viability of alloxan-treated cells. Intracellular ATP levels and glucose-stimulated insulin secretion were increased by RW to 2–3.5 times and 2–4 times, respectively, suggesting that RW enhances the glucose-sensitivity and glucose response of β-cells. The protective activity of RW was stable at 4 °C for over a month, but was lost by autoclaving. These results suggest that RW protects pancreatic β-cells from alloxan-induced cell damage by preventing alloxan-derived ROS generation. RW may be useful in preventing alloxan-induced type 1-diabetes mellitus. This revised version was published online in August 2006 with corrections to the Cover Date.     

Antiobesity and emetic effects of a short-length peptide YY analog and its PEGylated and alkylated derivatives

Neuropeptide Y2 receptor (Y2R) agonism is an important anorectic signal and a target of antiobesity drug discovery. Recently, we synthesized a short-length Y2R agonist, PYY-1119 (4-imidazolecarbonyl-[d-Hyp²⁴,Iva²⁵,Pya(4)²⁶,Cha^27,36,γMeLeu²⁸,Lys³⁰,Aib³¹]PYY(23–36), 1) as an antiobesity drug candidate. Compound 1 induced marked body weight loss in diet-induced obese (DIO) mice; however, 1 also induced severe vomiting in dogs at a lower dose than the minimum effective dose administered to DIO mice. The rapid absorption of 1 after subcutaneous administration caused the severe vomiting. Polyethylene glycol (PEG)- and alkyl-modified derivatives of 1 were synthesized to develop Y2R agonists with improved pharmacokinetic profiles, i.e., lower maximum plasma concentration (C_max) and longer time at maximum concentration (T_max). Compounds 5 and 10, modified with 20?kDa PEG at the N-terminus and eicosanedioic acid at the Lys³⁰ side chain of 1, respectively, showed high Y2R binding affinity and induced significant body weight reduction upon once-daily administration to DIO mice. Compounds 5 and 10, with their relatively low C_max and long T_max, partially attenuated emesis in dogs compared with 1. These results indicate that optimization of pharmacokinetic properties of Y2R agonists is an effective strategy to alleviate emesis induced by Y2R agonism.     

Optimal age-dependent sustainable harvesting of natural resource populations: Sustainability value

We studied the optimal age-dependent harvesting of a natural resource population that achieves a maximum income under the constraint of sustainability, i.e. the reproductive adults numbers must exceed a given minimum. The resource species is assumed to be semelparous (a single reproduction over a life). The economic value and natural mortality coefficient can vary with age. The optimal age-dependent harvesting under the sustainability constraint is obtained using Pontryagin’s maximum principle. The constraint of resource sustainability can be treated as an additional term measured in the same units as economic income. Specifically, three terms: (1) current harvesting value, (2) future harvesting value, and (3) sustainability value, are calculated for each age, and the resources should be harvested at the maximum rate when their current harvesting value is greater than the sum of future harvesting value and sustainability value, and should not be harvested otherwise. Numerical analyses of several cases demonstrated that the optimal harvesting schedule depends critically on the natural mortality coefficient and the functional form of the economic value of the resource.     

Methyl-4-phenylpyridinium (MPP(+))-evoked dopamine release from rat striatal slices: possible roles of voltage-dependent calcium channels and reverse dopamine transport.

We examined the properties of voltage-dependent Ca(2+) channels (VDCCs) mediating 1-methyl-4-phenylpyridinium (MPP(+))-evoked [3H]DA release from rat striatal slices. In some cases, the Ca(2+)-independent efflux of neurotransmitters is mediated by the high-affinity neurotransmitter-uptake systems. To determine whether such a mechanism might be involved in MPP(+)-evoked [3H]DA release. MPP(+) (1,10 and 100 microM) evoked the release of [3H]DA from rat striatal slices in a concentration-dependent manner. In the absence of Ca(2+), MPP(+) (10 and 100 microM)-evoked [3H]DA release was significantly decreased to approximately 50% of control (a physiological concentration of Ca(2+)). In the presence of Ca(2+), nomifensine (0.1,1 and 10 microM) dose-dependently and significantly inhibited the MPP(+)-evoked release of [3H]DA. Nomifensine (1 and 10 microM) also dose-dependently and significantly inhibited the MPP(+)-evoked release of [3H]DA under Ca(2+)-free conditions. MPP(+)-evoked [3H]DA release was partly inhibited by nicardipine (1 and 10 microM), an L-type Ca(2+) channel blocker. On the other hand, the N-type Ca(2+) channel blocker omega-conotoxin-GVIA (omega-CTx-GVIA) (1 and 3 microM) did not affect this release. omega-agatoxin-IVA (omega-Aga-IVA) at low concentrations (0.1 microM), which are sufficient to block P-type Ca(2+) channels alone, also had no effect. On the other hand, MPP(+)-evoked [3H]DA release was significantly decreased by high concentrations of omega-Aga-IVA (0.3 microM) that would inhibit Q-type Ca(2+) channels. In addition, application of the Q-type Ca(2+) channel blocker omega-conotoxin-MVIIC (omega-CTx-MVIIC) (0.3 and 1 microM) also significantly inhibited MPP(+)-evoked [3H]DA release. These results suggest that MPP(+)-evoked [3H]DA release from rat striatal slices is largely mediated by Q-type Ca(2+) channels, and the Ca(2+)-independent component is mediated by reversal of the DA transport system.     

Functional involvement of Tudor and dPRMT5 in the piRNA processing pathway in Drosophila germlines

In Drosophila, the PIWI proteins, Aubergine (Aub), AGO3, and Piwi are expressed in germlines and function in silencing transposons by associating with PIWI‐interacting RNAs (piRNAs). Recent studies show that PIWI proteins contain symmetric dimethyl‐arginines (sDMAs) and that dPRMT5/Capsuleen/DART5 is the modifying enzyme. Here, we show that Tudor (Tud), one of Tud domain‐containing proteins, associates with Aub and AGO3, specifically through their sDMA modifications and that these three proteins form heteromeric complexes. piRNA precursor‐like molecules are detected in these complexes. The expression levels of Aub and AGO3, along with their degree of sDMA modification, were not changed by tud mutations. However, the population of transposon‐derived piRNAs associated with Aub and AGO3 was altered by tud mutations, whereas the total amounts of small RNAs on Aub and AGO3 was increased. Loss of dprmt5 did not change the stability of Aub, but impaired its association with Tud and lowered piRNA association with Aub. Thus, in germline cells, piRNAs are quality‐controlled by dPRMT5 that modifies PIWI proteins, in tight association with Tud.     

Molecular and functional characterization of choline transporter in rat renal tubule epithelial NRK-52E cells

Homeostatic regulation of the plasma choline concentration depends on the effective functioning of a choline transporter in the kidney. However, the nature of the choline transport system in the kidney is poorly understood. In this study, we examined the molecular and functional characterization of choline uptake in the rat renal tubule epithelial cell line NRK-52E. Choline uptake was saturable and mediated by a single transport system, with an apparent Michaelis-Menten constant (K_m) of 16.5 μM and a maximal velocity (V_max) of 133.9 pmol/mg protein/min. The V_max value of choline uptake was strongly enhanced in the absence of Na⁺ without any change in K_m values. The increase in choline uptake under Na⁺-free conditions was inhibited by Na⁺/H⁺ exchanger (NHE) inhibitors. Choline uptake was inhibited by the choline uptake inhibitor hemicholinium-3 (HC-3) and organic cations, and was decreased by acidification of the extracellular medium and by intracellular alkalinization. Collapse of the plasma membrane H⁺ electrochemical gradient by a protonophore inhibited choline uptake. NRK-52E cells mainly express mRNA for choline transporter-like proteins (CTL1 and CTL2), and NHE1 and NHE8. CTL1 protein was recognized in both plasma membrane and mitochondria. CTL2 protein was mainly expressed in mitochondria. The biochemical and pharmacological data indicated that CTL1 is functionally expressed in NRK-52E cells and is responsible for choline uptake. This choline transport system uses a directed H⁺ gradient as a driving force, and its transport functions in co-operation with NHE8. Furthermore, the presence of CTL2 in mitochondria provides a potential site for the control of choline oxidation.     

Dual wavelength imaging allows analysis of membrane fusion of influenza virus inside cells

Influenza virus hemagglutinin (HA) is a determinant of virus infectivity. Therefore, it is important to determine whether HA of a new influenza virus, which can potentially cause pandemics, is functional against human cells. The novel imaging technique reported here allows rapid analysis of HA function by visualizing viral fusion inside cells. This imaging was designed to detect fusion changing the spectrum of the fluorescence-labeled virus. Using this imaging, we detected the fusion between a virus and a very small endosome that could not be detected previously, indicating that the imaging allows highly sensitive detection of viral fusion.     

Diversity of γ- glutamyl peptides and oligosaccharides,the “kokumi” taste enhancers,in seeds from soybean mini core collections

Soybeans (Glycine max (L,) Merr,) contain γ-glutamyl peptides and oligosaccharides, and these components play an important role in imparting the “kokumi” taste to foods. To gain insight into the genetic diversities and molecular mechanisms of accumulation of γ-glutamyl peptides and oligosaccharides in soybean, we measured the contents of these components using the Japan and World mini core collections. Similar to other previously reported traits, wide variations were detected among the accessions in the core collections with respect to the content of γ-glutamyl peptides and oligosaccharides. We found a positive relationship between the content of γ-glutamyl tyrosine and γ-glutamyl phenylalanine and between the content of raffinose and stachyose. Furthermore, there were unique accessions that included high levels of γ-glutamyl peptides and oligosaccharides. These accessions may be helpful in understanding the accumulation mechanism of γ-glutamyl peptides and oligosaccharides and to increase the “kokumi” taste components in soybean by performing a genetic analysis.